In histopathology, there are well known methods of examining biological materials such as tissue or cells at microscopic scales. Typical methods used by pathologists for analysing cells involve the preparation of tissue samples by sectioning and staining samples and then examining the stained samples under an optical microscope. Colouring tissue using stains or dyes is a technique commonly used in microscopy to enhance contrast in the microscopic image. Different cell types (or cell components) may be combined with stains of different colours so that they can be easily differentiated. This enables diagnosis of abnormalities or diseases for example.
The set of stains in combination with the method of applying these stains to tissue samples on microscope slides are known as staining protocols. The most widely used staining protocol uses haematoxylin and eosin stains (“H&E” staining). The colour produced by each tissue preparation depends on the type of tissue (or cell component), the tissue thickness and the strength of the stain used. The intensity of the staining depends upon each of the tissue thickness and the stain concentration. This difference in staining strength is important and may influence the diagnostic result a pathologist decides upon.
In modern laboratories it has become common practice to view a sample of cells with a digital camera attached to a microscope or to use a very high resolution scanner to reproduce digitally the appearance of the cells over a larger area. This enables the images to be shared between pathologists without the need for them to be looking down the same microscope at the same sample. A significant problem with this practice, however, is that the colour appearance of the cells has an additional variation added by the colour response of the microscope, digital camera or scanner and the colour monitor or printer being used to view the image, as each of these devices has its own colour response. A means to calibrate and to assess the colour accuracy of these systems is highly desirable.
The variation in colour appearance due to different image capture and reproduction equipment is a problem commonly encountered in colour imaging. This problem is normally addressed by using a standardised calibration process as defined by the International Color Consortium (ICC). This is a standardised way of translating digital values read by a device or equipment into colour measurements defined by the Commission Internationale de l'Eclairage (CIE). The ICC defines a file format which specifies a mathematical transform that can be used to convert the device colour values to colour values in an interchange space that uses device independent CIE colour coordinates. Thus a colour produced by one image capture device or reproduction device can be mapped onto another device such that to a typical person they appear to be the same colour. This file format is commonly referred to as an “ICC profile”.
In the graphic arts, an ICC profile is generated by producing matched pairs of values. The reproduction part of the process, i.e. by monitor display or printing, is common in the graphic arts and in a similar way ICC profiles can be used with the reproduction of microscopy images. Typically an input device such as a graphic arts scanner or a studio camera is calibrated or characterised using a colour calibration target. The calibration target typically contains a set of colour patches having a wide range of colours. The colour patches are imaged with the input device which usually produces three values for each colour patch, commonly called RGB, or red, green, blue triplets. The corresponding colour patches of the chart are then measured with a spectrophotometer or other suitable instrument which typically produces 32 sets of spectral reflectance or transmittance values across the visible spectrum. These spectral values can then be converted to CIE colour coordinates using the equations defined by the CIE, which are typically triplets such as CIELab or CIEXYZ. An ICC profile describes the mapping from the RGB values to the CIE colour values.
The colour patches are normally combined into a mosaic of patches referred to as a calibration chart. Typically this is a chart as defined by ISO 12641:1997 “Graphic technology—Prepress digital data exchange—Colour targets for input scanner calibration”, but there are other examples of such calibration charts such as the XRite Color Checker. It is normal for the calibration chart to contain the colours typically presented to the image capture system such as a microscope with an attached digital camera.
For graphic arts applications, these calibration charts are produced in every type of film substrate used in order to ensure that the spectral content of each patch is the same as the spectral content of the images being scanned. Otherwise, a phenomenon known as metamerism can cause the ICC profile to correctly calibrate the chart but to give different colours for the image being scanned. In the case of a digital microscope used in pathology this phenomenon can occur when the same colour on the chart and the stained cell have a different spectral content which produce the same CIE colour value but different imaged RGB values.
One approach to more accurately reproduce the spectral response of biological stains is disclosed by the present applicant in WO2013/186530. That patent publication discloses a method of forming an imaging calibration device by depositing and localising tissue stain material in regions of the device, such as “wells”, which are defined within a gasket placed on top of a glass slide.
Despite the methods and apparatus discussed in WO2013/186530, there remains a need to provide improved devices, and methods of their production, which can be used to assess the accuracy with which a digital microscope system is able to reproduce slides stained using a given staining protocol, together with a calibration system for digital microscopes that minimises the effect of metamerism.
It would in principle be possible to use standardised biological tissue samples to address these needs however there are a number of difficulties with this approach:    (a) it is difficult to produce standardised tissue and to produce sections of standard thickness,    (b) stained tissue samples exhibit significant variation in colour within a cell and this makes it difficult to obtain regions of uniform colour of a size that can be measured and    (c) tissue samples degrade with time resulting in significant colour shifts.
There is therefore a need to identify a non-tissue substrate that can be stained to produce the same colours as biological tissue samples and which may be readily manipulated so as to provide imaging calibration or reference devices for biological imaging systems.